Solubilization of Aminopteridines

Journal of Pharmaceutical Sciences

878 and Demuth E. Bey. 34 1309(19013. (3) Jao, E . - C . , i d S h c h u k i n a , M.,j.Gen.'Ch;m. U . S . S . R . , (2) Bamberger E

29, 992(1959). (4) Gabriel, S., and Meyer, R., Em., 14, 823(1881).

Keyphrases o-Azidobenzaldehyde-syn thesis 2-Substituted indazoles-synthesis

Palladium-on-carbon- hydrogenation Condensation reactions

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Solubilization of Aminopteridines By MILTON LAPIDUS Aqueous solutions of a series of aminopteridines (5-10 mg./ml.) stable at physiologic pH's were prepared either by salt formation with hydrochloric acid or by complex formation with 10 percent deoxyribonucleic acid. Deoxyribonucleic acid was superior t o ribonucleic acid, crystalline bovine serum albumin, human serum albumin fraction V, or gum acacia i n solubilizing 4,7-diamino-2 (p-chloropheny1)N-(2-diethylaminoethyl)-6-pteridinecarboxamidea t pH 7. Potentiometric ritration detected complex formation between deoxyribonucleic acid and the above aminopteridine. linear response was obtained in the concentration range of 2-9 mcg./ml. pH of Complete Solubility and Solubility Reversal-To 25 mg. of each of the aminopteridines suspended in 50 ml. of water increments of 1 N HC1 were added until the soluble end point was reached. The pH was monitored with a Beckman pH meter, and p H equilibrium was established after the addition of each increment of acid. Back titration with 0.1 N sodium hydroxide resulted in the end point of solubility reversal. pH Solubility Profile of Wy-4029-To a series of 500-mg. samples of Wy-4029 suspended in 20 ml. of water were added varying amounts of concentrated hydrochloric acid to obtain a constant pH of 3, 4, 5, 6 , or 7. The contents of each beaker was stirred for 1 hr. at 25". The mixtures were then filtered through a 0.45-p Millipore filter (the standard method subsequently used) aud the concentration of Wy-4029 in the filtrate was determined. Potentiometric Evidence of Complex FormationSolutions of Wy-4029 (50 mg./100 ml.), DNA-Na (1 Gm./100 ml.), and Wy-4029 (50 mg./100 ml.) EXPERIMENTAL plus DNA-Na (1 Gm./100 ml.) were adjusted to The aminopteridines studied were synthesized by p H 3.5 with 1 N hydrochloric acid and titrated Osdene et al. (6, 7). Deoxyribonucleic acid-sodium with 0.1 N sodium hydroxide, p H equilibrium being (DNA-Na), ribonucleic acid-sodium (RNA-Na), established after each addition of alkali. and crystalline bovine serum albumin (BSA) were Stability of Macromolecule-Aminopteridine Compurchased from Nutritional Biochemical Corp., plex-To 25 mg. of each of the aminopteridines was Cleveland, Ohio; human serum albumin (HA) frac- added 50 ml. of a 1% solution of DNA-Na (pH tion V from Pentex, Inc., Kankakee, Ill.; and USP 2.5), and the mixtures were triturated in a Pottergum acacia from S. B. Penick and Co., New York, Elvehjem homogenizer until soluble ( 5 min.). N. Y. The standard reagents used were the best Increments of 0.1 N sodium hydroxide were slowly grades commercially available. added, pH equilibrium being established after the Analysis of Wy-4029-Wy-4029 [4,7-diamino-2- addition of each increment. The pH at which ( p chlorophenyl) - N - ( 2 - diethylaminoethyl) - 6- turbidity occurred was considered the end point pteridinecarboxamide] in 0.1 N sulfuric acid fluo- of solubility reversal. resces a t 445 m p when activated a t 390 mp. Using Effect of Concentration and pH of Macromolecules the Aminco Bowman spectrophotofluorometer, a on Wy-4029 Solubility-To various concentrations of DNA-Na (pH 5.0), KNA-Ns (pli 2.8), HA Received October 27, 1967, from the Research Division, (pH 4.9), BSA (pH 5.1), and USP gum acacia Wyeth Laboratories, Inc., Philadelphia, PA 19101 Accepted for publication January 11, 1968. (pH 4.4) in 20 ml. of water was added an excess The author acknowledges lhe techuical assistance of Mr. (50-400 mg.) of Wy-4029. The mixtures were D. D. Austin.

course of preparing injectable solutions of a number of aminopteridines it was observed that very few in the series under investigation were appreciably soluble in water. The general lack of aqueous solubility of substituted pteridines has been reported as being due t o substituted polar groups (OH, SH, and NH1) (1). Pteridine (unsubstituted) was reported as being highly soluble (1 part in 7.5 parts of water at 20'). Although classic salt formation has been used successfully to solubilize aliphatic amines, this method has limited applicability t o weakly basic aminopteridines. The apparent acid solubility of triamterene (2,4,7-triamino-6-phenylpteridine) is reversed as the pH is raised to 7 (2). Complex formation has been reported to increase the aqueous solubility of small molecules (3-5). Speculation that deoxyribonucleic acid, ribonucleic acid, serum proteins, and gum acacia may form aminopteridine complexes with enhanced solubility characteristics stimulated this study.

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879

Vol. 57, No. 5, May 1968 triturated in a Potter-Elvehjem homogenizer for 5 min., stirred an additional 2 hr., adjusted t o p H 7, filtered, and the concentrations of Wy-4029 determined. Effect of Initial pH of the Macromolecule Solution on Solubility of Wy-4029-To 20 ml. of 5% solutions of DNA-Na, RNA-Na, gum acacia, and BSA adjusted with 1 N hydrochloric acid or 1 N sodium hydroxide to pH's varying from 2 to 8 was added an excess (100-400 mg.) of Wy-4029. The mixtures were triturated in a Potter-Elvehjem homogenizer for 5 min., stirred an additional 2 hr., adjusted to pH 7, filtered, and the concentrations of Wy-4029 determined. Preparation of Wy-4029-DNA Solution-This solution as prepared by adding 10 Gm. of DNA-Na to 90 ml. of water, adjusting to pH 3.5 with dilute hydrochloric acid, making up to 100 ml., and clarifying by centrifugation a t 6,000 r.p.m. for 15 min. Wy-4029 (1 Gm.) was added and complete solubility was attained by trituration in a Potter-Elvehjem glass homogenizer. Each prepared solution was vigorously stirred and slowly adjusted to pH 7 with dilute sodium hydroxide, then either sterile-filtered through a 0 . 4 5 ~ Millipore filter and stored in ampules or lyophilized and stored as a powder. This powder was readily reconstituted by the addition of water.

TADLE I-EFFECT OF DNA

ON

RESULTS AND DISCUSSION

The aminopteridines investigated were all solubilized by classic salt formation (0.5 mg./ml.) with 0.1 N hydrochloric acid. By allowing pH equilibrium after the addition of each increment of acid, it was possible to arrive a t the pH at which aqueous solubility was achieved. The compounds varied considerably in solubility pH (Table I). The iiidividual values probably reflect the relative basicity: the weaker this basicity, the greater the difficulty of forming classic salts and the lower the pH required for solubilization. Reversal of the apparent acid solubility of the aminopteridines, accomplished by the addition of sufficient 0.1 N sodium hydroxide, also occurred over a rather wide pH range (Table I). A number of aminopteridines remained soluble a t 0.5 mg./ml. and pH 7.4, and for these compounds it was predicted that higher concentrations would be stable. Solutions of aminopteridines (10 mg./ml.) were then prepared by solubilization with hydrochloric acid and adjustment to pH 7.4 with alkali (Table I, footnote 6).

To determine the effect of DNA on aminopteridine solubility, a 1% solution of DNA-Na (pH 2.5) coiltaining 0.5 mg./ml. of an aminopteridine in solution was titrated with 0.1 N sodium hydroxide until solubility reversal occurred. In every example the rever-

"EXTENDING" PH SOLUBILITY OF AMINOPTERIDINES NH,

WY-

R1

Rz

Ra

ReReversal versa1 Solof of D N A uble Salt SoluHCIa Salt, Solubility, bility, P 11 PH PH

*

1841':

-CONHCH?CHKH?NRti

-NHz

4.4

9.1

10.1

1843'

-CONHCHzCHzNEtz

-NHz

4.2

8.4

-

-NHz

4.4

5.4

9.U

351gd

-NH,

D

3648d

-CONHCHzCHCHzHMez

-NH2

3.9

7.7

9.0

3665'

-CONHCHL!HzCH?NEtz

-NHz

4.6

8.5

9.9

3873'

-CONHCHzCHzCHzNMe?

-NHz

5.2

9.1

9 8

-CONHCHzCHzNMez

--N H?

4.8

7.9

9.3

-CONHCHKHCHzNI?tz

-NHz

8..5

10.8

-

-CONHCH$2HtNRt2

-NHa

4.1

7.7

9.4

7.0

9.0

419CJ

-CONHC H?CH?NEtz

-NHz

4.3

(Conlinned on next page)

880

Journal of Pharmaceutical Sciences TABLE I-( Covctinued) R ___

P-

WY-

RI

R%

SolReversal versa1 uble of of DNA HCh Salt SoluSalt, Solubility, bility, PH PH PH

*

427(jC

-CONHCHCHZNHI

-NHI

5.2

>10.1

-

4437c

-CONHCHz(CHz)2CH?NMez

-NH?

7.6

9.2

-

-CON HCHCHZCHINF,~>

-NHz

4.4

8.5

9.6

5120'

-CONHCHzCH2NEtz

-NHCHzCH2NEtz

8.0

8.8

9.8

512lC

-CONHCHz(CHz)CH2NEt,

-NHs

4.2

9.3

10.3

5250'

-CONHCHz(CHz)aCHzNMeI

-NH2

3.6

8.5

10.5

52561

-CONHCH2CHzOMe

-NHCHzCHzOMe

2.5

6.1

6.9

5330'

-CONHCIIz(CHz)aCH2NMez

-NHz

4.9

8.4

10.0

5365d

-CONHCKz(CHz)CH?NEt?

-NH2

3.2

8.7

9.9

5588'

-CONHCII2CH?NRtr

-NHz

4.8

8.5

9.7

682Oe

-CONHCHzCHzNEta

-NHi

5.0

7.6

9.6

-CONHCHCHzNEtz

-NHs

4.6

8.3

-

--CONHCHs(CH2)zCHzNJvIez

-NHz

5.6

8.5

10.4

4Y23d

aMe

7037'

Me

7038'

Q CI

Aminopteridine, 25 mg./50 ml. of a 1% solution of DNA-Na. Solubilized by Aminopteridine, 25 mg./50 ml. water. Solubilized by Solubilized by complex formation, 5 mg./ml. in 10% DNA, pH 7.0. salt formation, 10 mg./ml. H 7 4. Solubilized by complex formation, 5 mg./ml. in 10% DNA, pH 6.5. complex formation, 10 m g . ~ m lin . 10% DNA. pH 7.4. @

*

sal occurred at a higher p H than had previously been determined for solutions prepared by classic salt formation (Table I). The 0.7-4.5 unit upward extension of the pH solubility range suggested the concomitant utility of complex formation by DNA as well as other macromolecules for preparing stable solutions of aminopteridines a t higher concentrations than was previously possible. As shown in Table I, some aminopteridines cannot be solubilized a t 10 mg./ml. and p H 7.4, probably because they are weakly basic and do not form stable hydrochlorides. Wy-4029, representative of this group, has limited solubility at p H 7 (Fig. 1). The concentration of Wy-4029 in solution was increased considerably by complex formation with macromolecules. Solutions of DNA-Na (pH 5), RNA-Na (pH 2.8), HA fraction V (pH 4.9), BSA (pH 5.1), and gum acacia (pH 4.4) at their respective natural pH's were effective in solubilizing Wy-4029 and in maintaining concentrations a t p K 7 previously not considered possible (Fig. 2). Since the p H of the macromolecule solution is a variable in determining the amount of aminopteridine solubilized, a pH-solubility profile

study of 5% solutions of DNA-Na, RNA-Na, BSA, and gum acacia was made. For each of the macromolecules an optimum pH for maximum solubility of Wy-4029 was determined (Fig. 3). The anticipated superiority of DNA-Na in solubilizing Wy-4029 was confirmed. The potentiometric evidence of complex formation between Wy-4029 and DNA-Na was provided by three distinct alkali titration curves (Fig. 4) (8). Complex formation existed up to approximately pH 9. DNA-Na maintained the solubility of Wy-4029 above the p H a t which solubility by salt formation failed. A solution of Wy-4029 solubilized in 10% DNANa a t p H 3.5 (10 mg./ml.) and then adjusted t o pH 7.4 was found to be stable at 4' and 25" for a minimum of 3 months. A lyophilized powder prepared from a freshly made solution was readily reconstituted in water. A toxicity evaluation of the aminopteridines and aminopteridine-DNA complexes (1 mg./mouse) demonstrated the protective effect of DNA in preventing skin lesions in mice at the subcutaneous site

Vol. 57, No. 5, May 1968

881

18-

I6 x

4

14-

92-

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# 6421

2

3

4

5

6

7

PH Fig. 1-pH

Solubility profile of Wy-4029 in presence of hydrochloric acid.

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Fig. 3-Solubility of Wy-4029 at pH 7 in 5% solution of each of four macromolecules as a functzon of the solubilization p H . , Key: O , DNA-Na; 0, R N A Na; A, gum acacza; and O , bovine serum albumin. lO.0r

2

6

10 14 18 22 26 MACROMOLECULE, %

30

.,

Fig. 2-Solubility at pH 7 of Wy-4029 complexed with each of jive macromolecules. Complexes of Wy-4029. Key: 0 , DNA-Na, (pH 5 ) ; . 0, R N A Na, (pH 2.8); human albumin fractaon V (pH 4.9); n, bovine serum albumin ( p H 5.1); and, A, gum acacia (pH 4.4). Wy-4029 was solubzlazed initially at the given natural p H (shown in parentheses) of each of the macromolecules. of injection. Dissection of the subcutaneous site failed to reveal the depot of aminopteridine usually found after injecting an aqueous solution. It seems reasonable to assume that transport of the aminopteridine away from the site of injection is accelerated when it is complexed with DNA. REFERENCES (I) Albert, A,, Brown, D. J., and Cheeseman, G . , J . Chem. SOL.,1952 4219. (2) Ditiert, L. W., Higuchi, T., and Reese, D. R.. J . Pharm. Sci., 5 3 , 1325(1964). (3) Dairghaday, W.H., Physiol. R e x , 39, 995(1959). (4) Riva, S. C., Biochem. Biophys. Res. Commun., 23, 606 (1966). ( 5 ) Bischoff, F.,and Pilhorn, H. R., J . Biol. Chem., 174,

RtX(1948) _ _ . ~

(6) Osdene, T. S . , Santilli, A. A., McCardle, L. E., and Rosenthale M. E., J . M e d . Chcm., 9 697(1966).

3.oL

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, , , , , , , , , , , , , , 8 10 12 14 16 18 20 0.1 N NaOH, ml.

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Keyphrases

1 Aminopteridines-solubilization Complex dines

formation-DNA-aminopteri-

Macromolecules-solubilizing effects pH-Solubility

profile

Toxicity-DNA-aminopteridines